Project Summary This proposal will test a model of DNA duplex recognition by the CRISPR-Cas12a nuclease (previously called ?Cpf1?). The goal is to understand the mechanistic basis leading to the high degree of DNA discrimination observed for Cas12a in the cell, and, in the long term, to enable better design of CRISPR variants with enhanced specificity for genome engineering. Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immunity mechanism used by bacteria and archaea to combat invading viruses and other mobile genetic elements. In both the type II CRISPR-Cas9 and the type V CRISPR-Cas12a systems, an effector complex comprised of a single protein activated by CRISPR-encoded small RNA(s) (crRNA) recognizes and cleaves double-stranded DNAs at specific sites. The groundbreaking 2013 discovery that Cas9 can be programmed with engineered small RNAs to efficiently edit eukaryotic genomes sparked a revolution in genome engineering that is still rapidly unfolding. Cas12a, which was first characterized at the end of 2015, has been successfully used for genome editing. Cas12a shows stronger capability for DNA discrimination than Cas9, and its ability to process the pre-crRNA allows more efficient multiplex genome editing. With these features, Cas12a holds great potentials for development of better CRISPR-based tools. As with Cas9, a key step in target acquisition in Cas12a is the unwinding of the DNA duplex to form a stable R-loop structure, in which the crRNA guide-segment is base-paired with the target-strand of the DNA. Studies have shown that Cas12a more stringently discriminates against mismatches in the RNA/DNA hybrid than Cas9, and the mismatch tolerance patterns significantly differ between Cas12a and Cas9. However, the mechanism that gives rise to the higher specificity in Cas12a is unknown. Based on available literature and our preliminary data, we propose a two-stage unwinding model for Cas12a. In collaboration with the laboratory of Feng Zhang (Broad Institute of MIT and Harvard), we will test this model by probing the unwinding state of various DNA segments as Cas12a binds a target duplex and cleaves each of the strands. The studies will leverage our recently published work demonstrating the use of a biophysical method ? site-directed spin labeling ? to directly detect Cas9-mediated DNA unwinding. The spin labeling method will be combined with a fluorescence unwinding assay and detailed kinetic and thermodynamic analyses of DNA cleavage and binding. We expect that data obtained will provide a definitive assessment of the two-stage unwinding model. If proven correct, the model will account for Cas12a?s enhanced and distinct mismatch discrimination pattern, and the mechanistic information will guide further development of CRISPR-based genome editing technology.